Cerebral edema (and its counterpart in the spinal cord) is a major contributor to the damage caused by stroke, traumatic brain injury, traumatic spinal-cord injury, and bacterial meningitis. Stroke alone is the third leading cause of death in the USA and the leading cause of disability. Unfortunately, only a few treatment options are available for cerebral and spinal-cord edema, and these are of limited efficacy. Central in the development of cerebral and spinal-cord edema is the H2O channel aquaporin-4 (AQP4) in the astrocytic endfeet that envelope CNS blood vessels at the blood-brain barrier (BBB). AQP4 is upregulated by ischemia, traumatic brain injury, and a diverse array of other CNS pathologies associated with inflammation. But, paradoxically, AQP4-null mice do substantially better than their wild-type counterparts in experimental models of ischemic stroke, retinal ischemia, and bacterial meningitis, making AQP4 an attractive drug target. A unique aspect of the present proposal is the recognition that the AQPs are bifunctional proteins that are not only permeable to water, but also to the dissolved gases O2 and CO2. Indeed, the upregulation of AQP4 in the aforementioned pathologies may have the beneficial effect of maximizing O2 permeability across the BBB, with the side effect of increasing osmotic water permeability and predisposing to cerebral edema. The long-term goal of Aeromics is to develop a drug that treats cerebral edema by blocking the water permeability of AQP4, leaving the gas permeability intact. The goals of this SBIR application are to: (1) Develop high-throughput screens (HTSs) for inhibitors of the water permeability of AQP4, using optical methods to assess the consequences of osmotically induced cell swelling. (2) Establish counterscreens for water and gas permeabilities, using biophysical approaches founded on electrophysiology and optics. These counterscreens will identify: (a) false positives from the HTS, (b) compounds that block gas as well as H2O permeability, and (c) compounds that reduce the water permeability of AQPs other than AQP4. (3) Perform pilot high-throughput screens using the MicroSource GenPlus 960 and the Maybridge 20k libraries, and identify some core structures. The proposed work will prepare Aeromics for a Phase II study, in which the HTS would be greatly expanded to identify several core structures that would be developed into leads for new drugs to treat cerebral edema. Swelling of the brain (cerebral edema) and spinal cord contributes in a major way to the damage caused by stroke, traumatic brain injury, traumatic spinal-cord injury, and bacterial meningitis. Unfortunately, only a few treatment options are available for cerebral and spinal-cord edema, and these are of limited efficacy. The goal of this project is to develop the technology to identify new drugs for treating cerebral edema while maintaining the delivery of oxygen to the brain.